Antifungal phenylethylene

ABSTRACT

The antifungal and cancer cell growth inhibitory activities of 1-(3′,4′,5′-trimethoxyphenyl)-2-nitro-ethylene (TMPN) were examined. TMPN was fungicidal for the majority of 132 reference strains and clinical isolates tested, including those resistant to fluconazole, ketoconazole, amphotericin B or flucytosine. Minimum fungicidal concentration/minimum inhibitory concentration (MFC/MIC) ratios were ≦2 for 96% of  Cryptococcus neoformans  clinical isolates and 71% of  Candida albicans  clinical isolates. TMPN was fungicidal for a variety of other basidiomycetes, endomycetes and hyphomycetes, and its activity was unaffected by alterations in media pH. TMPN was slightly cytotoxic for murine and human cancer cell lines (GI 50 =1-4 μg/ml), and weakly inhibited mammalian tubulin polymerization (IC 50 =0.60 μg/ml). TMPN may also be used as a biochemical probe for tubulin and fungal dimorphism studies.

RELATED APPLICATION

This application claims the priority of U.S. Provisional Application No.60/343,067, filed Dec. 22, 2001, entitled “Antifingal Phenethylene,”which is incorporated herein by reference.

GOVERNMENT INTEREST

This invention was funded in part by the NIH OIGCA44344-01-011. TheUnited States Government may have certain rights in this invention.

INTRODUCTION

This invention relates to phenethylene compounds having antifungalactivity. More particularly this invention relates to the development ofa phenethylene having significant antifungal activity, namely1-(3′,4,′5′-trimethoxyphenyl)-2-nitro-ethylene, which may also be usedas a biochemical probe for tubulin and fungal dimorphism study.

BACKGROUND

The major classes of antifungal drugs available for clinical use are themacrolide polyenes, fluoropyrimidines, azoles and theallylamines/thiocarbamates [1]. These agents are limited by toxicity,fungistatic mechanisms, narrow activity spectra and/or drug resistance[1]. The limited selection of effective antifungals, combined with theemergence of previously uncommon fungal pathogens [2] and an increasingpopulation of immunocompromised patients, has resulted in a criticalneed for new antifungal agents. The development of compounds of novelstructural class that have a fungicidal mechanism and a broad spectrumof activity will likely have the greatest impact on the current crisis.

BRIEF DESCRIPTION OF THE INVENTION

A recent review of the antifungal actions of antineoplastic agentsconcluded that antineoplastic agents and their derivatives are anexcellent resource for the discovery of novel antifungal targets andagents [3]. A lead in vitro antifungal compound with cancer cell lineinhibitory activity, 1-(3′,4′,′5′-trimethoxyphenyl)-2-nitro-ethylene(TMPN) was discovered. TMPN was synthesized as part of astructure/activity study of trimethoxybenzene antitubulin compounds likepodophyllotoxin.

The antifingal and cancer cell growth inhibitory activities TMPN wereexamined. TMPN was fungicidal for the majority of 132 reference strainsand clinical isolates tested, including those resistant to fluconazole,ketoconazole, amphotericin B or flucytosine. Minimum fungicidalconcentration/minimum inhibitory concentration (MFC/MIC) ratios were ≦2for 96% of Cryptococcus neoformans clinical isolates and 71% of Candidaalbicans clinical isolates. TMPN was fungicidal for a variety of otherbasidiomycetes, endomycetes and hyphomycetes, and its activity wasunaffected by alterations in media pH. The frequency of fungalspontaneous mutations to resistance was <10⁻⁶.

Kill curve analyses confirmed the fungicidal action of TMPN, anddemonstrated that killing was concentration- and time-dependent. Atsub-MIC exposure to TMPN, C. albicans did not exhibit yeast/hyphaeswitching. TMPN was slightly cytotoxic for murine and human cancer celllines (GI₅₀=1-4 μg/ml), and weakly inhibited mammalian tubulinpolymerization (IC₅₀=0.60 μg/ml). The in vitro profile of TMPN warrantsits development both as an in vivo antimicrobial for superficial andcutaneous mycoses, and as a biochemical probe for tubulin and fungaldimorphism study.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Structure of 1-(3′,4′,5′-trimethoxyphenyl)-2-nitro-ethylene.

FIG. 2. Kill curves for C. neoformans ATCC 90112 (A) and C. albicansATCC 90028 (13) with indicated multiples of the1-(3′,4′,5′-trimethoxyphenyl)-2-nitroethylene MIC. Results are means±thestandard errors of the means.

FIG. 3. Percentage of C. albicans ATCC 90028 cells with buds (solidlines) or hyphal extensions (dotted lines): control cells treated withDMSO (squares); cells treated with one quarter times the TMPN MIC(upside down triangles); cells treated with one half times the TMPN MIC(circles). The results are presented as means±standard errors of themeans.

FIG. 4. Morphological characteristics of control and TMPN-treated C.albicans ATCC 90028 observed with video-enhanced DIC optics. Scalebar=10 μm. In panel a, yeast growth (arrowhead) and hyphal growth(arrows) morphologies were observed under control growth conditions. Inpanel b, only yeast growth (arrowheads) morphology was observed incultures treated with one half times the TMPN MIC.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The major classes of antifungal drugs available for clinical use are themacrolide polyenes, fluoropyrimidines, azoles and theallylamines/thiocarbamates [1]. Toxicity, fungistatic mechanisms, narrowactivity spectra and/or drug resistance [1] limit these agents. Thelimited selection of effective antifungals, combined with the emergenceof previously uncommon fungal pathogens [2] and an increasing populationof immunocompromised patients, has resulted in a critical need for newantifungal agents. The development of compounds of novel structuralclass that have a fungicidal mechanism and a broad spectrum of activitywill likely have the greatest impact on the current crisis.

A recent review of the antifungal actions of antineoplastic agentsconcluded that antineoplastic agents and their derivatives are anexcellent resource for the discovery of novel antifungal targets andagents [3]. The present invention demonstrates the in vitro developmentof an antifungal compound with cancer cell line inhibitory activity,1-(3′, 4′,5′-trimethoxyphenyl)-2-nitro-ethylene (TMPN) who's structuralformula is depicted in FIG. 1.

TMPN was synthesized as part of a structure/activity study oftrimethoxybenzene antitubulin compounds like podophyllotoxin. The effectof TMPN on a variety of cell types was investigated. The in vitroprofile of TMPN warrants its development as an antimicrobial forsuperficial and cutaneous mycoses, and as a biochemical probe fortubulin and fungal dimorphism studies. TMPN was synthesized aspreviously described in the art. TMPN was reconstituted in a smallvolume of sterile dimethylsulfoxide (DMSO) and diluted in theappropriate media immediately prior to susceptibility experiments.

Reference strains were obtained from the American Type CultureCollection (Rockville, Md.) or Presque Isle Cultures (Presque Isle,Pa.). Strains were maintained by single colony transfer on nutrient agarat 35° C. (exceptions were Neisseria gonorrhoeae on gonococcal typingagar [5] at 37° C. with 5% CO₂, and Streptococcus pneumoniae on trypticsoy agar with 5% sheep blood at 37° C. with 5% CO₂.

Nonduplicate clinical isolates were obtained at the University ofVirginia Health System. Fluconazole-resistant clinical isolates [Jessup,C. J., Wallace, T. L. & Ghannoum, M. A. (1997) Evaluation of antifungalactivity of nyotran against various pathogenic fungi. Poster #F-88.Toronto: 37th Interscience Conference on Antimicrobial Agents andChemotherapy] were provided by the Center for Medical Mycology, CaseWestern Reserve University. Reference strains were obtained from theAmerican Type Culture Collection.

Most yeast strains were maintained by single colony transfer onSabouraud Dextrose Agar (SDA), pH 5.6 at 35° C. Cryptococcus albidus, C.laurentii and C. uniguttulatus (#66033) were maintained on SDA, pH 6.6at 25° C., Filobasidium uniguttulatum, Kluyveroinyces spp., Trichosporonspp., Blastoschizomyces capitatus, Epiderinophyton floccosum andPaeciloinyces lilacinus on Emmon's modification of SDA at 30° C., and C.uniguttulatus (#34143) and C. ater on Yeast Morphology (YM) agar at 25°C. Rhizopus spp. and Aspergillus spp. were maintained on Potato DextroseAgar (PDA) slants at 35° C.

Antimicrobial activity was assayed by National Committee for ClinicalLaboratory Standards (NCCLS) disk susceptibility tests. Isolatedcolonies from overnight cultures were suspended and diluted asrecommended to yield approximately 1-2×10⁸ cfu/ml, and 50 μl of thispreparation immediately spread on agar plates. NCCLS recommended agarmedia [5] were used for S. pneumoniae and N. gonorrhoeae, andMueller-Hinton agar for all other bacteria. Yeast strains were tested onSDA. Excess moisture was absorbed for 10 min prior to application of 6mm paper discs containing two-fold dilutions of TMPN in sterile DMSO.The MIC was defined as the lowest drug concentration resulting in aclear zone of growth inhibition around the disc after 18 h (allorganisms except Micrococcus, Candida, Cryptococcus) or 42 h(Micrococcus, Candida, Cryptococcus).

The antibacterial activity of TMPN was also assessed by the NCCLS brothmacrodilution assay [6]. Isolated colonies from overnight cultures weresuspended and diluted as recommended to yield final inocula ofapproximately 5×10⁵ cfu/ml. Tests were performed in sterile plastictubes (12 by 75 mm) containing twofold dilutions of TMPN in gonococcaltyping broth (Neisseria), Mueller Hinton II (MHII) (cation adjusted)broth containing 3% lysed horse blood (Streptococcus) or MHII broth (allother bacteria). One tube was left drug-free (but contained anequivalent volume of DMSO) for a turbidity control. Tubes were incubatedwithout agitation at 37° C. with 5% CO₂ (Streptococcus, Neisseria), orat 35° C. (remaining bacteria). MICs were determined after 24 h for allbacteria except Micrococcus, which was read at 48 h. The MIC was definedas the lowest concentration of drug that inhibited all visible growth ofthe test organism (optically clear).

TMPN was screened against yeasts by the broth macrodilution assayaccording to the NCCLS [7]. Yeasts were suspended and diluted asrecommended to yield final inocula ranging from 0.5-2.5×10³ cfu/ml.Tests were performed in sterile 12 by 75 mm plastic tubes containingtwo-fold dilutions of TMPN in 0.165 M morpholinepropanesulfonic acidbuffered RPMI 1640 medium (pH 7.0). One tube was again left drug free(but contained an equivalent volume of DMSO) for a turbidity control.Tubes were incubated without agitation at the appropriate temperature(see Fungal Strains section above). MICs were determined after 72 h forCryptococcus and after 48 h for other yeast genera. The MIC was definedas the lowest concentration of TMPN that inhibited all visible growth ofthe test organism (optically clear).

Susceptibility testing of filamentous fungi was also conducted. Brothmacrodilution susceptibility testing of P. lilacinus, Rhizopus spp. andAspergillus spp. was performed in accordance with a proposedstandardized procedure [8] with slight modification. To induce conidiumand sporangiospore formation, fungi were grown on PDA slants at 35° C.for 6 days (P. lilacinus, Rhizopus spp., all Aspergillus species exceptA. nidulans) or 3 days (A. nidulans). Fungal slants were covered withsterile 0.85% NaCl (P. lilacinus, Rhizopus spp., all Aspergillus speciesexcept A. nidulans) or 0.05% Tween 80 (A. nidulans), and suspensionswere made by gently probing the colonies with the tip of a sterilePasteur pipette. The resulting mixture of hyphal fragments and conidiaor sporangiospores was withdrawn and transferred to a sterile clearmicrocentrifuge tube, and heavy particles were allowed to settle for 10min. The upper homogenous suspension was transferred to a sterilemicrocentrifuge tube, vortexed for 15 s, adjustedspectrophotometrically, and diluted in sterile 0.165 M MOPS-bufferedRPMI medium, pH 7.0, to yield final inocula ranging from 0.5-2.5×10³cfu/ml. Susceptibility to TMPN was then determined by brothmacrodilution as described above for yeast isolates. MICs were readafter 48 h The MIC was defined as the lowest concentration of TMPN thatinhibited all visible growth of the test organism (optically clear).

Minimum fungicidal concentrations (MFCs) were determined by subculturing0.1 ml from each tube with no visible growth in the MIC brothmacrodilution series onto the appropriate drug-free plates (see FungalStrains section above). The plates were incubated for 48 h, and the MFCwas defined as the lowest drug concentration that completely inhibitedgrowth on plates.

Possible host effects were also evaluated. Broth macrodilution assayswere performed with RPMI medium prepared at pH 5, pH 6, and pH 7, and inRPMI medium with and without 50% normal human serum (Lampire BiologicalLabs). The pH experiments were performed twice on separate days.

Determination of the frequency of occurrence of spontaneous mutants wasperformed as previously described [9]. Overnight cultures of Cneoformans (ATCC 90112), C. albicans (ATCC 90028) and Trichosporon inkin(ATCC 18020) were diluted to an OD_(530 nm=)0.3. 0.1 ml of eachpreparation was spread onto SDA plates containing four or eight timesthe broth macrodilution MIC of TMPN. The starting inoculum for eachorganism was also diluted and plated onto drug-free SDA plates fordetermination of cfu/ml. After a 48 h incubation at the appropriatetemperature (see Fungal Strains section above), the number of colonieson drug-supplemented SDA was counted. The frequency of occurrence ofspontaneous mutants was calculated by dividing the number of colonies ondrug-containing plates by the number of colonies in the inoculum. Whenno colonies were visualized on drug-containing plates, the calculationwas (<) 1 colony divided by the number of colonies in the inoculum.

Time—kill studies were also performed. The proposed standardizedprocedures of Klepser et al [10] were followed. Overnight cultures of C.neoformans (ATCC 90112) and C. albicans (ATCC 90028) in pH 7.0MOPS-buffered RPMI 1640 medium were inoculated into the same mediumcontaining multiples of the broth macrodilution MIC of TMPN or anequivalent volume of DMSO. Cultures were shaken at 35° C., and aliquotsaseptically removed at various times for dilution plating. In addition,100 μl aliquots were plated directly from drug-treated flasks at eachtime point. Thus, the detection limit in these experiments was 10cfu/ml. Standard errors of the means were calculated from at least twoexperiments.

The mechanism of action of TMPN was investigated microscopically,Candida albicans (ATCC 90028) cultures were exposed to one quarter orone half times the broth macrodilution MIC of TMPN in DMSO, or anequivalent concentration of DMSO for controls, until late-log phase.Cells were examined using an Axioscope microscope (Carl Zeiss,Thornwood, N.Y.) equipped with standard differential interferencecontrast (DIC) using a Plan-Neofluar 100×/1.3 (oil immersion) objective.The microscope was coupled to a C24007-07 (imaging tube camera type)video camera, via a 4×extension tube (Carl Zeiss), and an analog controlunit (Hammamatsu Photonic Systems Corp., Bridgewater, N.J.). Real-timedigital contrast enhancement was done with an Argus 10 Image Processor(Hammamatsu). Single frame images were digitized directly or fromvideotaped sequences using a Sony UP-5600MD video/digital printer (SonyElectronics, Inc., Montvale, N.J.) and prepared for printing inPhotoshop 5.0 (Adobe Systems, Mountain View, Calif.). Final images wereprinted with a NP-1600M Medical Color Printer (Codonics, Inc.,Middleburg Heights, Ohio). To determine the proportion of cellsexhibiting hyphal or yeast growth morphology, four areas with 200 cellseach were counted on two separate days (total of 1600 cells), and thestandard errors of the means calculated.

An investigation TMPN's in vitro antineoplastic activity was alsoconducted. This investigation included an analysis of both cell growth,and of the effects of TMPN upon tubulin. Inhibition of cancer cellgrowth was assessed using the Sulforhodamine B assay as previouslydescribed [11]. Briefly, cells in 5% fetal calf serum/RPMI-1640 wereinoculated into 96 well plates, incubated 24 h and 10-fold dilutions ofTMPN added. After a 48 h incubation, plates were fixed withtrichloroacetic acid, washed, stained with Sulforhodamine B and readwith an automated microtiter plate reader.

Electrophoretically homogeneous bovine brain tubulin [12] was used instudies to evaluate the effects of TMPN on in vitro tubulinpolymerization and the binding of [¹³H]colchicine (Dupont-NEN) totubulin. These studies were performed as described previously [13]. Inthe polymerization assay, varying drug concentrations were added to 1mg/ml tubulin to determine the amount of drug that would inhibit theextent of the reaction by 50% (20 min incubation at 30° C.) (IC₅₀value). In the colchicine binding assay, the effect of varying drugconcentrations on the binding of 2 μg/ml colchicine to 100 μg/ml tubulinwas measured after 10 min at 37° C. (control reaction about 50%complete).

The results obtained may be summarized as follows. In disk diffusionassays, the synthetic compound TMPN inhibited the growth of yeasts andcertain bacteria, primarily gram-positive bacteria as shown in Table 1.However, in broth macrodilution assays, MICs for all bacteria were >64μg/ml [single exception was N. gonorrhoeae with an MIC of 4 μg/ml. Brothmacrodilution assays revealed that TMPN had broad-spectrum antifungalactivity. (Table 2) MFC/MIC ratios were <2 for 96% of C. neoformansclinical isolates, 71% of C. albicans clinical isolates and 70% of C.krusei clinical isolates. TABLE 1 Antimicrobial activities of1-(3′,4′,5′-trimethoxyphenyl)- 2-nitro-ethylene in the disk diffusionassay Organism MIC (μg/disk) Staphylococcus aureus ATCC 29213 3.12-6.25Staphylococcus epidermidis Presque Isle 4653  50-100 Enterococcusfaecalis ATCC 29212  50-100 Streptococcus pneumoniae ATCC 6303 3.12-6.25Micrococcus luteus Presque Isle 456  50-100 Bacillus subtilis PresqueIsle 620  50-100 Stenotrophomonas maltophilia ATCC 13637 >100Pseudomonas aeruginosa Presque Isle 99 >100 Escherichia coli ATCC25922 >100 Neisseria gonorrhoeae ATCC 49226 0.39-0.78 Enterobactercloacae ATCC 13047 >100 Klebsiella pneumoniae Presque Isle 344 >100Proteus vulgaris Presque Isle 365 12.5-25   Cryptococcus neoformans ATCC90112 0.78-1.56 Candida albicans ATCC 90028 6.25-12.5

TABLE 2 Broth macrodilution MICs and MFCs of1-(3′,4′,5′-trimethoxyphenyl)- 2-nitro-ethylene for reference strainsand clinical isolates MIC (μg/ml) MFC (μg/ml) Organism (no. of strains)Range 50%^(a) 90%^(a) Range 50%^(b) 90%^(b) Fluconazole-resistantCryptococcus neoformans (4) 4-8  4-16 C. neoformans (24)  2-16 4 8  4-328 16 C. neoformans ATCC 90112 2 4 C. neoformans ATCC 66031 2 4 C.neoformans ATCC 14116 8 16 C. neoformans ATCC 32045 4 4 C. ater ATCC14247 8 16 C. uniguttulatus ATCC 66033 16 32 C. uniguttulatus ATCC 341438 16 C. laurentii ATCC 66036 16 32 C. laurentii ATCC 34142 32 32 C.laurentii ATCC 18803 16 32 C. albidus ATCC 66030 8 16 C. albidus ATCC40666 8 32 C. albidus ATCC 34140 32 32 Filobasidium uniguttulatum ATCC24227 0.5 2 Candida albicans (7)  4-32    4->64 Ketoconazole-resistantC. albicans ATCC 64124 16 32 Flucytosine-resistant C. albicans ATCC32354 16 16 C. albicans ATCC 90028 32 32 C. albicans ATCC 10231 8 8 C.albicans ATCC 14053 16 16 C. albicans ATCC 60193 16 16 C. parapsilosis(10) 16-32   32->64 C. parapsilosis ATCC 22019 16 32 AmphotericinB-resistant C. lusitaniae ATCC 42720 16 16 C. glabrata (8)  8-32  16->64 C. glabrata ATCC 90030 4 16 C. glabrata ATCC 2001 8 16 C.guilliermondii (9)   64->64 >64 C. krusei (10)  8-16 8 16 16-64 32 32 C.rugosa (7)  1-64    8->64 C. tropicalis (9) 32-64   32->64 C. utilisATCC 22023 8 8 C. utilis ATCC 9226 4 8 C. viswanathii ATCC 22981 32 >64Rhodotorula mucilaginosa ATCC 9449 4 8 Kluyveromyces marxianus ATCC365534 4 4 K. apiculate ATCC 9774 1 2 Trichosporon cutaneum ATCC 28592 48 T. inkin ATCC 18020 8 16 T. asahii ATCC 20039 8 16 T. mucoides ATCC90046 16 32 T. ovoides ATCC 90040 16 32 Blastoschizomyces capitalus ATCC10663 8 32 Epidermophyton floccosum ATCC 52066 2 ND Paecilomyceslilacinus (1) 32 64 Rhizopus oligosporus ATCC 22959 8 >64 R. nigracans(ASU culture collection) 32 >64 Aspergillus fumigatus ATCC 96918 8 >64A. nidulans strain FGSC4^(b) 8 16 A. flavus (ASU culture collection)16 >64 A. niger (ASU culture collection) 16 64^(a)50% and 90%, MICs at which 50 and 90% of the strains, respectively,are inhibited.^(b)50% and 90%, MFCs at which 50% and 90% of the strains, respectively,are killed.

When data for all 60 Candida spp. clinical isolates were combined, 47%had MFC/MIC ratios ≦2, and 60% had MFC/MIC ratios ≦4. MICs and MFCs wereidentical or differed by no more than a single 2-fold dilution whenbroth macrodilution assays were performed at pH 5, pH 6 and pH 7 (Table3). The compound was not active against all species in the presence of50% human serum, and serum inactivation did not appear to be due toserum albumin binding or serum agglutinins. The frequency of occurrenceof single-step resistant mutants at four times the broth macrodilutionMIC was ≦10⁻⁶ for the three strains tested, C. neoformans (ATCC 90112),C. albicans (ATCC 90028) and T. inkin (ATCC 18020). FIG. 2 summarizesthe time-kill curves for C. neoformans (ATCC 90112) (FIG. 2A) and C.albicans (ATCC 90028) (FIG. 2B). For C. neoformans, time to 99.9% killwas between 4 and 6 h at the MIC. For C. albicans, time to 99.9% killwas between 2 and 4 h at four times the MIC. TABLE 3 Effect of pH, humanserum or bovine serum albumin on MICs and MFCs of1-(3′,4′,5′-trimethoxyphenyl)-2-nitro-ethylene MIC (MFC) OrganismTreatment in μg/ml Cryptococcus neoformans pH 5  2 (4), 1 (2)^(a). ATCC90112 pH 6  4 (4), 4 (4) pH 7  2 (4), 2 (4) no serum  2 (4) 50% humanserum >64 no bovine serum albumin  2 (4) 20 μg/ml bovine serum albumin 4 (4) 40 μg/ml bovine serum albumin  4 (4) Candida albicans pH 5 32(32), 32 (32) ATCC 90028 pH 6 32 (32), 32 (32) pH 7 32 (32), 32 (32) noserum 32 (32) 50% human serum >64 no bovine serum albumin 32 (32) 20μg/ml bovine serum albumin 32 (32) 40 μg/ml bovine serum albumin 16 (32)Trichosporon inkin ATCC 18020 pH 5  4 (8), 4 (8) pH6  8 (16), 8 (16) pH7 8 (16), 8 (16) no serum  8 (16) 50% human serum >64 Aspergillusfumigatus pH 5  4 (32), 8 (>64) ATCC 96918 pH 6  4 (32), 8 (>64) pH 7  4(32), 8 (>64) no serum  8 (16) 50% human serum 16 (>64)^(a)repeat experiment

Video-enhanced DIC optics were used to investigate possiblemorphological alterations in drug-treated C. albicans (ATCC 90028).Cultures were exposed to varying concentrations of TMPN or an equivalentconcentration of DMSO (controls), and samples removed late log-phase formicroscopy (FIGS. 3,4). From 2-8 h, cultures treated with DMSO alone hadapproximately the same number of cells with buds as cells with hyphalextensions. Although C. albicans grew at the same rate as controls whenexposed to one half times the TMPN MIC, cells with hyphal extensionswere not observed in one half times the MIC-treated cultures. Hyphaewere rarely seen in one quarter times the MIC-treated cultures, andremained <20 μm in length.

TMPN inhibited the growth of the murine P388 lymphocytic leukemia cellline and six human cancer cell lines, (Table 4) with GI₅₀ values rangingfrom 1.1-4.1 μg/ml. For inhibition of tubulin polymerization, TMPN wascompared with the potent colchicine binding site agent combretastatinA-4. TMPN had an IC₅₀=0.60±0.07 (S.D.) μg/ml for inhibition of theextent of assembly (20 min incubation at 30° C.) versus 0.32+0.02 μg/mlfor combretastatin A-4. Combretastatin A-4 at 1.6 μg/ml (5 μM) inhibited[³H]colchicine binding to tubulin by 98+1%, while TMPN at 1.2 μg/ml (5μM) was minimally inhibitory (13±0.5%). However, when the TMPNconcentration was raised to 12 μg/ml (50 uM), there was 69±0.5%inhibition. TABLE 4 Inhibition of murine P388 lymphocytic leukemia andhuman cancer cell line growth by1-(3′,4′,5′-trimethoxyphenyl)-2-nitro-ethylene Cell line GI₅₀ ^(a)(μg/ml) P388 leukemia 4.15 Pancreas BXPC-3 1.6 Ovarian OVCAR-3 1.8 CNSSF-295 2.0 Lung-NSC NCI-H460 1.4 Colon KM20L2 1.4 Prostate DU-145 1.1^(a)GI₅₀ , inhibition of 50% of cell growth.

Based upon the foregoing observations, these compositions are believeduseful in the treatment of one or more fungal infections, such asAspergillosis, Candidiasis or thrush, internal infections such ascryptococcosis, epidermal infections, infections caused by antibioticresistant fungi and the like. Similar fungal infections are enumeratedin the AMA Home Medical Encyclopedia published by Random House, Inc.1989.

The dosage administered will be dependent upon the identity of thefungus; the location of the fungal infection; the type of host involved;the nature of concurrent treatment, if any; and the frequency oftreatment specified.

Illustratively, dosage levels of the administered active ingredientsare: intravenous, 0.1 to about 200 .mu.g/kg; orally, 5 to about 1000mu.g/kg of host body weight. Expressed in terms of concentration, anactive ingredient can be present in the compositions of the presentinvention for localized use about the cutis, intranasally,pharyngolaryngeally, bronchially, intravaginally, or ocularly in aconcentration of from about 0.01 to about 50% w/w of the composition;preferably about 1 to about 20% w/w of the composition; and forparenteral use in a concentration of from about 0.05 to about 50% w/v ofthe composition and preferably from about 5 to about 20% w/v.

The compositions of the present invention are preferably presented foradministration to humans and animals in salves and ointments for topicalapplication although unit dosage forms, such as tablets, capsules,pills, powders, suppositories, sterile parenteral solutions orsuspensions, sterile non-parenteral solutions or suspensions, lozengesand the like, containing suitable quantities of an active ingredient.

For oral administration either solid or fluid unit dosage forms can beprepared. Powders are prepared quite simply by comminuting the activeingredient to a suitably fine size and mixing with a similarlycomminuted diluent. The diluent can be an edible carbohydrate materialsuch as lactose or starch. Advantageously, a sweetening agent or sugaris present as well as a flavoring oil. Preparing a powder mixture ashereinbefore described and filling into formed gelatin sheaths producescapsules. Advantageously, as an adjuvant to the filling operation, alubricant such as talc, magnesium stearate, calcium stearate and thelike is added to the powder mixture before the filling operation.

Soft gelatin capsules are prepared by machine encapsulation of a slurryof active ingredients with an acceptable vegetable oil, light liquidpetrolatum or other inert oil or triglyceride.

Tablets are made by preparing a powder mixture, granulating or slugging,adding a lubricant and pressing into tablets. The powder mixture isprepared by mixing an active ingredient, suitably comminuted, with adiluent or base such as starch, lactose, kaolin, dicalcium phosphate andthe like. The powder mixture can be granulated by wetting with a bindersuch as corn syrup, gelatin solution, methylcellulose solution or acaciamucilage and forcing through a screen. As an alternative to granulating,the powder mixture can be slugged, i.e., run through the tablet machineand the resulting imperfectly formed tablets broken into pieces (slugs).The slugs can be lubricated to prevent sticking to the tablet-formingdies by means of the addition of stearic acid, a stearic salt, talc ormineral oil. The lubricated mixture is then compressed into tablets.

Advantageously, the tablet can be provided with a protective coatingconsisting of a sealing coat or enteric coat of shellac, a coating ofsugar and methylcellulose and polish coating of carnauba wax.

Fluid unit dosage forms for oral administration such as in syrups,elixirs and suspensions can be prepared wherein each teaspoonful ofcomposition contains a predetermined amount of an active ingredient foradministration. The water-soluble forms can be dissolved in an aqueousvehicle together with sugar, flavoring agents and preservatives to forma syrup. An elixir is prepared by using a hydroalcoholic vehicle withsuitable sweeteners together with a flavoring agent. Suspensions can beprepared of the insoluble forms with a suitable vehicle with the aid ofa suspending agent such as acacia, tragacanth, methylcellulose and thelike.

For parenteral administration, fluid unit dosage forms are preparedutilizing an active ingredient and a sterile vehicle, water beingpreferred. The active ingredient, depending on the form andconcentration used, can be either suspended or dissolved in the vehicle.In preparing solutions the active ingredient can be dissolved in asuitable vehicle for injection and filter sterilized before filling intoa suitable vial or ampule and sealing. Advantageously, adjuvants such asa local anesthetic, preservative and buffering agents can be dissolvedin the vehicle.

Parenteral suspensions are prepared in substantially the same mannerexcept that an active ingredient is suspended in the vehicle instead ofbeing dissolved and sterilization cannot be accomplished by filtration.The active ingredient can be sterilized by exposure to ethylene oxidebefore suspending in the sterile vehicle. Advantageously, a surfactantor wetting agent is included in the composition to facilitate uniformdistribution of the active ingredient.

In addition to oral and parenteral administration, the vaginal routescan be utilized particularly by means of a suppository. A vehicle whichhas a melting point at about body temperature or one that is readilysoluble can be utilized. For example, cocoa butter and variouspolyethylene glycols (Carbowaxes) can serve as the vehicle.

For use as aerosols, the active ingredients can be packaged in apressurized aerosol container together with a gaseous or liquefiedpropellant, for example, dichlorodifluoromethane, carbon dioxide,nitrogen, propane, and the like, with the usual adjuvants such ascosolvents and wetting agents, as may be necessary or desirable.

In a preferred practice for the treatment of dermatological fingi, theactive ingredient will be delivered to the site as an ointment or salvethat will comprise water and oil emulsion as the principal carrier.Other conventional ingredients, when conditions and aesthetics dictate,include petrolatum and mineral oil, lipophilic solubilizers such aspolyethylene glycol, carbowax, moisturizers such as lanolin andfragrance.

The term “unit dosage form” as used in the specification and claimsrefers to physically discrete units suitable as unitary dosages forhuman and animal subjects, each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effectin association with the required pharmaceutical diluent, carrier orvehicle. The specifications for the novel unit dosage forms of thisinvention are dictated by and are directly dependent on (a) the uniquecharacteristics of the active material and the particular therapeuticeffect to be achieved, and (b) the limitation inherent in the art ofcompounding such an active material for therapeutic use in humans, asdisclosed in this specification, these being features of the presentinvention. Examples of suitable unit dosage forms in accord with thisinvention are tablets, capsules, troches, suppositories, powder packets,wafers, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampules,vials, segregated multiples of any of the foregoing, and other forms asherein described.

The active ingredient to be employed as an antifingal agent can beeasily prepared in such unit dosage form with the employment ofpharmaceutical materials which themselves are available in the art andcan be prepared by established procedures. The following preparationsare illustrative of the preparation of the unit dosage forms of thepresent invention, and not as a limitation thereof. Several dosage formswere prepared embodying the present invention. They are shown in thefollowing examples in which the notation “active ingredient” signifiesTMPN, or a close homolouge, inclusive.

Composition “A”

Hard-Gelatin Capsules

One thousand two-piece hard gelatin capsules for oral use, each capsulecontaining 200 .mu.g of an active ingredient are prepared from thefollowing types and amounts of ingredients: Active ingredient,micronized 200 g Corn Starch  20 g Talc  20 g Magnesium stearate  2 g

The active ingredient, finely divided by means of an air micronizer, isadded to the other finely powdered ingredients, mixed thoroughly andthen encapsulated in the usual manner. The foregoing capsules are usefulfor treating a fungal disease by the oral administration of one or twocapsules one to four times a day.

Using the procedure above, capsules are similarly prepared containing anactive ingredient in 50, 250 and 500 mu.g amounts by substituting 50.mu.g, 250 .mu.g and 500 .mu.g of an active ingredient for the 200 .mu.gused above.

Composition “B”

Soft Gelatin Capsules

One-piece soft gelatin capsules for oral use, each containing 200 .mu.gof an active ingredient, finely divided by means of an air micronizer,are prepared by first suspending the compound in 0.5 ml of corn oil torender the material capsulatable and then encapsulating in the abovemanner.

The foregoing capsules are useful for treating a fungal disease by theoral administration of one or two capsules one to four times a day.

Composition “C”

Tablets

One thousand tablets, each containing 200 .mu.g of an active ingredient,are prepared from the following types and amounts of ingredients: Activeingredient, micronized 200 g Lactose 300 g Corn starch  50 g Magnesiumstearate  4 g Light liquid petrolatum  5 g

The active ingredient, finely divided by means of an air micronizer, isadded to the other ingredients and then thoroughly mixed and slugged.The slugs are broken down by forcing them through a Number Sixteenscreen. The resulting granules are then compressed into tablets, eachtablet containing 200 .mu.g of the active ingredient.

The foregoing tablets are useful for treating a fungal disease by theoral administration of one or two tablets one to four times a day. Usingthe procedure above, tablets are similarly prepared containing an activeingredient in 250 .mu.g and 100 .mu.g amounts by substituting 250 .mu.gand 100 .mu.g of an active ingredient for the 200 .mu.g used above.

Composition “D”

Oral Suspension

One liter of an aqueous suspension for oral use, containing in eachteaspoonful (5 ml) dose, 50 .mu.g of an active ingredient, is preparedfrom the following types and amounts of ingredients: Active ingredient,micronized 10 g Citric acid 2 g Benzoic acid 1 g Sucrose 790 gTragacanth 5 g Lemon Oil 2 g Deionized water, q.s. 1000 ml

The citric acid, benzoic acid, sucrose, tragacanth and lemon oil aredispersed in sufficient water to make 850 ml of suspension. The activeingredient, finely divided by means of an air micronizer, is stirredinto the syrup unit uniformly distributed. Sufficient water is added tomake 1000 ml. The composition so prepared is useful for treating afungal disease at a dose of 1 teaspoonful (15 ml) three times a day.

Composition “E”

Parenteral Product

One liter of a sterile aqueous suspension for parenteral injection,containing 30. mu.g of an active ingredient in each milliliter fortreating a fungal disease, is prepared from the following types andamounts of ingredients: Active ingredient, micronized 30 g POLYSORBATE80 5 g Methylparaben 2.5 g Propylparaben 0.17 g Water for injection,q.s. 1000 mi.

All the ingredients, except the active ingredient, are dissolved in thewater and the solution sterilized by filtration. To the sterile solutionis added the sterilized active ingredient, finely divided by means of anair micronizer, and the final suspension is filled into sterile vialsand the vials sealed. The composition so prepared is useful for treatinga fungal disease at a dose of 1 milliliter (1 ml) three times a day.

Composition “F”

Vaginal Suppository

One thousand suppositories, each weighing 2.5 g and containing 200 .mu.gof an active ingredient are prepared from the following types andamounts of ingredients: Active ingredient, micronized   15 g Propyleneglycol   150 g Polyethylene glycol #4000, q.s. 2,500 g

The active ingredient is finely divided by means of an air micronizerand added to the propylene glycol and the mixture passed through acolloid mill until uniformly dispersed. The polyethylene glycol ismelted and the propylene glycol dispersion is added slowly withstirring. The suspension is poured into unchilled molds at 40.degree. C.The composition is allowed to cool and solidify and then removed fromthe mold and each suppository foil wrapped. The foregoing suppositoriesare inserted vaginally for treating candidiasis (thrush).

Composition “G”

Intranasal Suspension

One liter of a sterile aqueous suspension for intranasal instillation,containing 20 .mu.g of an active ingredient in each milliliter, isprepared from the following types and amounts of ingredients: Activeingredient, micronized 15 g POLYSORBATE 80 5 g Methylparaben 2.5 gPropylparaben 0.17 g Delonized water, q.s. 1000 ml.

All the ingredients, except the active ingredient, are dissolved in thewater and the solution sterilized by filtration. To the sterile solutionis added the sterilized active ingredient, finely divided by means of anair micronizer, and the final suspension is aseptically filled intosterile containers.

The composition so prepared is useful for treating a fungal disease, byintranasal instillation of 0.2 to 0.5 ml given one to four times perday. An active ingredient can also be present in the undiluted pure formfor use locally about the cutis, intranasally, pharyngolaryngeally,bronchially, or orally.

Composition “H”

Powder

Five grams of an active ingredient in bulk form is finely divided bymeans of an air micronizer. The micronized powder is placed in ashaker-type container. The foregoing composition is useful for treatinga fungal disease, at localized sites by applying a powder one to fourtimes per day.

Composition “I”

Oral Powder

One hundred grams of an active ingredient in bulk form are finelydivided by means of an air micronizer. The micronized powder is dividedinto individual doses of 200 .mu.g and packaged. The foregoing powdersare useful for treating a fungal disease, by the oral administration ofone or two powders suspended in a glass of water, one to four times perday.

Composition “J”

Insufflation

One hundred grams of an active ingredient in bulk form are finelydivided by means of an air micronizer. The foregoing composition isuseful for treating a fungal disease, by the inhalation of 300 .mu.g oneto four times a day.

Composition “K”

Ointment

One hundred grams of an active ingredient in bulk form are finelydivided by means of an air micronizer. The micronized powder is themadmixed into a water and oil emulsion with the addition of suitablemoisturizers and fragrances as desired. The foregoing ointment is usefulfor treating a fungal disease by one topical application of the ointmenton the affected area as needed, preferably at least twice a day.

From the foregoing, it becomes readily apparent that a new and usefulantifingal agent and new and useful antifungal preparations have beenherein described and illustrated which fulfill the aforestated object ina remarkably unexpected fashion. It is, of course, understood that suchmodifications, alterations and adaptations as will readily occur to theartisan confronted with this disclosure are intended within the spiritof the present invention which is limited only by the scope of theclaims appended hereto

1. A method of treating a host afflicted by a fungi (this includes yeastand filamentous forms) by administering an effective amount of1-(3′,4,′5′-trimethoxyphenyl)-2-nitro-ethylene thereto.
 2. The methodaccording to claim 1 wherein said administration is systemic.
 3. Themethod according to claim 1 wherein said administration is topical.
 4. Amethod of using an effective amount of1-(3′,4,′5′-trimethoxyphenyl)-2-nitro-ethylene as a biochemical probe.5. A method according to claim 4 wherein said probe is used toinvestigate fungal dimorphism.